The present invention generally relates to compositions, and methods for use thereof, effective in regulating inflammatory platelet and endothelial stimulatory and coagulopathic responses by modulating the activity of the C5b-9 complex of the human plasma complement system by genetic manipulation.
The complement system is a complex interaction of plasma proteins and membrane cofactors which act in a multi-step, multi-protein cascade sequence in conjunction with other immunological systems of the body to provide immunity from intrusion of foreign cells. Complement proteins represent up to about 10% of globulins in normal serum of man and other vertebrates.
The classic complement pathway involves an initial antibody recognition of, and binding to, an antigenic site (SA) on a target cell. This surface bound antibody subsequently reacts with the first component of complement, C1q, forming a C1-antibody complex with Ca++, C1r, and C1s which is proteolytically active. C1s cleaves C2 and C4 into active components, C2a and C4a. The C4b,2a complex is an active protease called C3 convertase, and acts to cleave C3 into C3a and C3b. C3b forms a complex with C4b,2a to produce C4b,2a,3b, which cleaves C5 into C5a and C5b. C5b combines with C6. The C5b,6 complex combines with C7 to form the ternary complex C5b,6,7. The C5b,6, 7 complex binds C8 at the surface of the cell, which may develop functional membrane lesions and undergo slow lysis. Upon binding of C9 to the C8 molecules in the C5b,6,7,8 complex, lysis of bacteria and other foreign cells is rapidly accelerated.
Recently, the C5b-9 proteins of the human plasma complement system have been implicated in non-lytic stimulatory responses from certain human vascular and blood cells. The capacity of C5b-9 to modify membrane permeability and to selectively alter ion conductance is thought to elicit these non-lytic responses from human cells. In the case of human blood platelets and vascular endothelium, assembly of the C5b-9 complex initiates a transient and reversible depolarization of the plasma membrane potential, a rise in cytosolic Ca2+, metabolic conversion of arachidonate to thromboxane or prostacyclin, and the activation of intracellular protein kinases. In addition, human platelets exposed to C5b-9 undergo shape changes, secretory fusion of intracellular storage granules with plasma membrane, and the vesiculation of membrane components from the cell surface. Human endothelial cells exposed to the human C5b-9 proteins secrete high molecular weight multimers of the platelet adhesion protein, von Willibrand Factor (vWF), and the intracellular granule membrane protein, GMP140, is translocated from the Weible-Palade body to the endothelial surface. High molecular weight multimers of vWF have been implicated in the pathogenesis of vaso-occlusive platelet adherence to endothelium and cell surface GMP140 has been implicated in the adherence of inflammatory leukocytes to endothelium.
These effects of complement proteins C5b-9 on platelet and endothelial cells alter the normal regulation of the enzymes of the plasma coagulation system at these cell surfaces. For example, the generation of platelet membrane microparticles by vesiculation results in the exposure of membrane binding sites for coagulation factor Va. Binding of factor Va to these membrane microparticle sites initiates assembly of the prothrombinase enzyme complex. This complex in turn accelerates coagulation factor Xa activation of prothrombin to thrombin which promotes plasma clotting. Similarly, C5b-9 binding to the endothelial cell results in the exposure of plasma membrane receptors for the prothrombinase complex, thereby accelerating the generation of thrombin from prothrombin at the endothelial surface.
This interaction between components of the complement and coagulation systems at the surface of blood platelets and endothelium can generate inflammatory and chemotactic peptides at sites of vascular thrombus formation and may contribute to the altered hemostasis associated with immune disease states. In addition, immune reactions affecting blood platelets and endothelium can lead to platelet aggregation, the secretion of proteolytic enzymes and vasoactive amines from platelet storage granules, and increase adherence of platelets and leukocytes to the endothelial lining of blood vessels.
It has been demonstrated that membrane-uptake of C3b and C5b-9 proteins can occur spontaneously during incubation of platelets in citrated plasma. Complement activation can also occur during blood collection as a result of exposure to plastic surfaces supporting the C3-convertase reaction. While the implications of complement activation during blood collection and in vitro storage for transfusion have not been directly addressed it is, nevertheless, known that plasma levels of coagulation factors V and VIII rapidly decline in stored platelet concentrates at a rate considerably faster than their decay in cell-free plasma, suggesting consumptive loss. Further, platelet collection and storage is associated with an increase in vesicular plasma membrane microparticles, a product of C5b-9 initiated platelet secretion. These physiological and enzymatic changes greatly reduce the potential shelf life of stored platelets, particularly platelet-rich plasma concentrates used for transfusions, which is generally only 72 hours at best. Furthermore, this interaction of activated C5b-9, platelets, and coagulation factors in stored platelet concentrates will adversely affect the hemostatic effectiveness of these units when infused.
In vitro human organ and tissue storage and survival of the transplanted graft is also adversely affected by the spontaneous activation of the complement system, resulting in membrane insertion of the C5b-9 proteins into vascular endothelium. Activation of C5 to C5a and C5b has been shown to be catalyzed by plastics and other synthetic membranes required to maintain perfusion of vascular beds during in vitro tissue and organ storage. In addition, membrane deposition of C5b-9 in vivo has been implicated in the acute rejection of transplanted tissue due to immune activation of the recipient's plasma complement system against the endothelial cells within the donor's organ.
Assembly of the C5b-9 complex is normally limited in plasma by the amount of C5b generated by proteolysis of C5 to its biologically-active fragments C5b and C5a. In addition to plasmin and other plasma or cell-derived proteases, two enzymes of the complement system can cleave C5 to C5a and C5b, the membrane-stabilized enzyme complexes C4b2a and C3bBb (C5- convertases). The activity of these two enzymes is normally inhibited on the surface of human blood and vascular membranes by the plasma membrane proteins, "membrane cofactor protein" (CD46), described by Lublin and Atkinson, Current Topics Microbiol. Immunol. 153:123 (1989) and "decay-accelerating factor" (CD55), Medof, et al., J. Exp. Med. 160:1558 (1984).
Platelet and endothelial cell activation by C5b-9 also has ramifications in autoimmune disorders and other disease states. The importance of spontaneous complement activation and the resulting exposure of platelets and endothelium to activated C5b-9 to the evolution of vaso-occlusive disease is underscored by consideration that a) leukocyte infiltration of the subendothelium, which is known to occur in regions of atheromatous degeneration and suggests localized generation of C5a at the vessel wall, is potentially catalyzed by adherent platelets and b) local intravascular complement activation resulting in membrane deposition of C5b-9 complexes accompanies coronary vessel occlusion and may affect the ultimate extent of myocardial damage associated with infarction.
There is now considerable evidence that the human erythrocyte membrane as well as the plasma membranes of other human blood cells and vascular endothelium are normally protected from these effects of complement by cell-surface proteins that specifically inhibit activation of the C5b-9 pore upon C9 binding to membrane C5b-8, as reported by Holguin, M. H., et al., J. Clin. Invest. 84, 7-17 (1989); Sims, P. J., et al., J. Biol. Chem. 264, 19228-19235 (1989); Davies, A., et al., J. Exp. Med. 170, 637-654 (1989); Roilins, S. A., and Sims, P. J. J. Immunol. 144, 3478-3483 (1990); and Hamilton, K. K., et al., Blood 76, 2572-2577 (1990). Plasma membrane constituents reported to exhibit this activity include homologous restriction factor (HRF) (C8-binding protein), with an apparent molecular mass of 65 kDa, as described by Zalman, L. S., et al., Proc. Natl. Acad. Sci., U.S.A. 83, 6975-6979 (1986) and Schonermark, S., et al., J. Immunol. 136, 1772-1776 (1986), and the leukocyte antigen CD59, a glycoprotein with an apparent molecular mass of 18-21 kDa, described by Sugita, Y., et al., J. Biochem. (Tokyo) 104, 633-637 (1988); Holguin, M. H., et al., (1989); Sims, P. J., et al., (1989); Davies, A., (1989); Rollins, S. A., and Sims, P. J. (1990); and Hamilton, K. K., et al., (1990). Accumulated evidence suggest that these two proteins exhibit quite similar properties, including the following: (1) both HRF and CD59 are tethered to the cell surface by a glycolipid anchor, and are deleted from the membranes of the most hemolytically sensitive erythrocytes that arise in the stem cell disorder paroxysmal nocturnal hemoglobinuria; (2) the activity of both inhibitors is species-restricted, showing selectivity for C8 and C9 that are derived from homologous (i.e. human) serum; and (3) both HRF and CD59 appear to function by inhibiting the activation of C9, decreasing the incorporation of C9 into the membrane C5b-9 complex, and limiting propagation of the C9 homopolymer.
In U.S. Pat. No. 5,135,916, Sims and Wiedmer disclose compositions and methods for use thereof relating to polypeptides having the ability to act as an inhibitor of complement C5b-9 complex activity. The compositions contain CD59, an 18 kDa protein found on the surface of human erythrocytes, active derivatives or fragments thereof which act to inhibit the activity of C5b-9, anti-idiotypic antibodies mimicking the action of the inhibitor proteins or antibodies against C7 or C9 which block the formation of the C5b-9 complex. The compositions can be used in vitro to inhibit C5b-9 related stimulatory responses of platelets and vascular endothelium of perfused organs and tissues, thereby preventing the C5b-9 initiated cell necrosis or stimulated secretion of proteolytic enzymes and the exposure of the procoagulant membrane receptors during collection and in vitro storage. In one variation of this embodiment, the vascular endothelium of organs and tissues to be transplanted are treated with these compositions to protect these cells from complement activation after transplantation. In another embodiment, immune disease states are treated by administering an effective amount of a C5b-9 inhibitor to suppress C5b-9 mediated platelet activation in vivo. Also disclosed are methods for the production of isolated polypeptides that are able to suppress complement C5b-9 mediated platelet and endothelial cell activation.
In the disease paroxysmal nocturnal hemoglobinuria, the red cell that is most sensitive to complement-mediated cytolysis is normally deficient in both CD55 (decay accelerating factor, the membrane inhibitor of the C3/C5-convertase) and in CD59 (the inhibitor of C5b-9). Although CD46, CD55, and CD59, serve as inhibitors of complement activation, there is now considerable evidence that CD59 provides the most effective protection from the cytolytic and cell-stimulatory effects of complement by specificcally inhibiting the activation of C9 into a membrane pore-forming structure. The deletion of CD59 from the plasma membrane renders erythrocytes highly susceptible to lysis by human plasma, an effect that is not observed when only CD55 (decay-accelerating factor) is deficient. Furthermore, an isolated deficiency of decay-accelerating factor (CD55) does not result in hemolytic disease, as reported by Lin, et al., Transfusion 28:427-429 (1988) and Telen, et al., J. Exp. Med. 167:1993-1998 (1988). By contrast, all of the clinical manifestation of severe paroxysmal nocturnal hemoglobinuria, including intravascular hemolysis and cerebral infarction, were observed in a patient that exhibited an isolated defect in CD59 expression (due to a mutation in the CD59 gene) with normal expression of decay-accelerating factor (CD55), as reported by Yamashina, et al., N. E. J. Med. 323:1184-1189 (1990).
Subsequent to U.S. Ser. No. 07/365,199, a cDNA encoding CD59 was reported by Sawada, et al., Nucleic Acids Res. 17(16), 6728 (submitted Jul. 25, 1989). cDNA encoding CD59 has also been cloned from human T-cell leukemia (YT) and human erythroleukemia (K562) cell lines, and CD59 antigen transiently expressed in COS cells, as reported by Philbrick, W. M., et al., Eur. J. Immunol. 20, 87-92 (1990). Walsh, et al., Eur. J. Immunol. 21:847-850 (1991), demonstrated expression of CD59 antigen with complement-inhibitory activity in rat cells transfected with cDNA for CD59.
It is an object of the present invention to provide a means and method for the modulation and inhibition of complement C5b-9 mediated platelet and endothelial cell activation in vivo and in vitro by genetic modification of cells to be transplanted or infused.
It is a further object of the present invention to provide a means and method for increasing the survival and therapeutic efficacy of platelets and tissues or organs collected and stored in vitro by genetic manipulation of the cells.